Stimulation of an immune response is dependent upon the presence of antigens recognised as foreign by the host immune system. The discovery of the existence of tumour associated antigens has now raised the possibility of using a host's immune system to intervene in tumour growth. Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.
Certain elements of the cellular immune response are capable of specifically recognising and destroying tumour cells. The isolation of cytotoxic T-cells (CTL) from tumour-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in natural immune defences against cancer (Cheever et al., Annals N.Y. Acad. Sci. 1993 690:101-112; Zeh H J, et al.; J. Immunol. 1999, 162(2):989-94). CD8-positive T-cells (TCD8+) in particular, which recognise Class I molecules of the major histocompatibility complex (MHC)-bearing peptides of usually 8 to 10 amino acid residues derived from proteins or defect ribosomal products (DRIPS) (Schubert U, et al., Nature 2000; 404(6779):770-774) located in the cytosol, play an important role in this response. The MHC-molecules of a human are also designated as human leukocyte-antigens (HLA).
There are two classes of MHC-molecules: MHC class I molecules that can be found on most cells having a nucleus that present peptides that result from proteolytic cleavage of endogenous proteins, DRIPS, and larger peptides. MHC class II molecules can be found predominantly on professional antigen presenting cells (APCs), and present peptides of exogenous proteins that are taken up by APCs during the course of endocytosis, and are subsequently processed (Cresswell P., Annu Rev. Immunol. 1994; 12:259-93). Complexes of peptide and MHC class I molecules are recognised by CD8-positive cytotoxic T-lymphocytes bearing the appropriate TCR, and complexes of peptide and MHC class II molecules are recognised by CD4-positive-helper-T-cells bearing the appropriate TCR. It is well known that the TCR, the peptide and the MHC are thereby abundant in a stoichiometric amount of 1:1:1.
CD4-positive helper T-cells play an important role in orchestrating the effector functions of anti-tumour T-cell responses. For this reason, the identification of CD4-positive T-cell epitopes derived from tumour associated antigens (TAA) may be of great importance for the development of pharmaceutical products for triggering anti-tumour immune responses (Kobayashi, H., et al., 2002. Clin. Cancer Res. 8:3219-3225; Gnjatic, S., et al., 2003. Proc. Natl. Acad. Sci. U.S.A. 100(15):8862-7). CD4+ T cells can lead to locally increased levels of IFNγ, a critical requirement of interferon gamma-mediated angiostasis for tumour rejection by CD8+ T cells (Qin Z, et al., Cancer Res. 2003 J; 63(14):4095-4100).
In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system, especially professional antigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells, macrophages, dendritic cells. In tumour patients, cells of the tumour have surprisingly been found to express MHC class II molecules (Dengjel J, et al., Clin Cancer Res. 2006; 12:4163-4170).
It was shown in mammalian animal models, e.g., mice, that even in the absence of CTL effector cells (i.e., CD8-positive T lymphocytes), CD4-positive T-cells are sufficient for inhibiting visualization of tumours via inhibition of angiogenesis by secretion of interferon-gamma (IFNγ) (Qin, Z. et al., 2000. Immunity. 12:677-686). Additionally, it was shown that CD4-positive T-cells recognizing peptides from tumour-associated antigens presented by HLA class II molecules can counteract tumour progression via the induction of antibody (Ab) responses (Kennedy, R. C., et al., Cancer Res. 63:1040-1045). In contrast to tumour-associated peptides binding to HLA class I molecules, only a small number of class II ligands of TAA have been described so far. See generally, the syfpeithi database listing known MHC ligands and peptide motifs and Cancer Immunity, the Journal of Academy of Cancer Immunology.
Since the constitutive expression of HLA class II molecules is usually limited to cells of the immune system (Mach, B., et al., 1996. Annu Rev. Immunol. 14:301-331), the possibility of isolating class II peptides directly from primary tumours was not considered possible. However, Dengjel et al. were recently successful in identifying a number of MHC Class II epitopes directly from tumours (See EP 04 023 546.7, EP 05 019 254.1; Dengjel J, et al., Clin Cancer Res. 2006; 12:4163-4170).
For a peptide to trigger (elicit) a cellular immune response, it must bind to an MHC-molecule. This process is dependent on the allele of the MHC-molecule and specific polymorphisms of the amino acid sequence of the peptide. MHC-class-I-binding peptides are usually 8-10 amino acid residues in length and usually contain two conserved residues (“anchors”) in their sequence that interact with the corresponding binding groove of the MHC-molecule. In this way each MHC allele has a “binding motif” determining which peptides can bind specifically to the binding groove (Rammensee H. G., et al, Chapman & 1998 Hall MHC Ligands and Peptide Motifs).
In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class 1 molecules expressed by tumour cells, they also have to be recognized by T-cells bearing specific T-cell receptors (TCR).
The antigens that are recognised by the tumour specific cytotoxic T-lymphocytes, that is, their epitopes, can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc., which are up-regulated in cells of the respective tumour. Furthermore, tumour associated antigens, for example, can also be unique to tumour cells, for example as products of mutated genes or from alternative open reading frames (ORFs), or from protein splicing (Vigneron N, et al., Science 2004 Apr. 23; 304 (5670):587-90). Another important class of tumour associated antigens are tissue-specific antigens, such as CT (“cancer testis”)-antigens that are expressed in different kinds of tumours and in healthy tissue of the testis.
Various tumour associated antigens have been identified. Further, much research effort has been spent to identify additional tumour associated antigens. Some groups of tumour associated antigens, also referred to in the art as tumour specific antigens, are tissue specific. Examples include, but are not limited to, tyrosinase for melanoma, PSA and PSMA for prostate cancer and chromosomal cross-overs such as bcr/abl in lymphomas. However, many tumour associated antigens identified to date occur in multiple tumour types, and some, such as oncogenic proteins and/or tumour suppressor genes (tumour suppressor genes are, for example reviewed for renal cancer in Linehan W M, et al., J. Urol. 2003 December; 170(6 Pt 1): 2163-72), which actually cause the transformation event, occur in nearly all tumour types. For example, normal cellular proteins that control cell growth and differentiation, such as p53 (which is an example for a tumour suppressor gene), ras, c-met, myc, pRB, VHL, and HER-2/neu, can accumulate mutations resulting in up-regulation of expression of these gene products thereby making them oncogenic (McCartey et al. Cancer Research 1998 15:58 2601-5; Disis et al. Ciba Found. Symp. 1994 187:198-211). These mutant proteins can also be a target of a tumour specific immune response in multiple types of cancer.
For proteins to be recognised by cytotoxic T-lymphocytes as tumour-specific or -associated antigens, and for them to be used in a therapy, particular prerequisites must be fulfilled. The antigen should be expressed mainly by tumour cells and not, or in comparably small amounts, by normal healthy tissues. It is furthermore desirable, that the respective antigen is not only present in a type of tumour, but also in high concentrations (i.e. copy numbers of the respective peptide per cell). Tumour-specific and tumour-associated antigens are often derived from proteins directly involved in transformation of a normal cell to a tumour cell due to a function e.g. in cell cycle control or apoptosis. Additionally, downstream targets of the proteins directly causative for a transformation may be upregulated and thus may be indirectly tumour-associated. Such indirectly tumour-associated antigens may also be targets of a vaccination approach (Singh-Jasuja H., et al., Cancer Immunol. Immunoether. 2004 March; 453 (3): 187-95). In both cases it is essential to have epitopes in the amino acid sequence of the antigen, since such peptide (“immunogenic peptide”) that is derived from a tumour associated antigen should lead to an in vitro or in vivo T-cell-response.
Basically, any peptide able to bind a MHC molecule may function as a T-cell epitope. A prerequisite for the induction of an in vitro or in vivo T-cell-response is the presence of a T-cell with a corresponding TCR and the absence of immunological tolerance for this particular epitope.
Therefore, TAAs are a starting point for the development of a tumour vaccine. The methods for identifying and characterizing the TAAs are based on the use of CTL that can be isolated from patients or healthy subjects, or they are based on the generation of differential transcription profiles or differential peptide expression patterns between tumours and normal tissues (Lemmel C., et al., Nat. Biotechnol. 2004 April; 22(4):450-4; T. Weinschenk, et al., Cancer Res. 62 (20):5818-5827, 2002.).
However, the identification of genes overexpressed in tumour tissues or human tumour cell lines, or selectively expressed in such tissues or cell lines, does not provide precise information as to the use of the antigens transcribed from these genes in an immune therapy. This is because only an individual subpopulation of epitopes of these antigens are suitable for such an application since a T-cell with a corresponding TCR has to be present and immunological tolerance for this particular epitope needs to be absent or minimal. It is therefore important to select only those peptides from overexpressed or selectively expressed proteins that are presented in connection with MHC molecules against which a functional T-cell can be found. Such a functional T-cell is defined as a T-cell that upon stimulation with a specific antigen can be clonally expanded and is able to execute effector functions (“effector T-cell”).
T-helper cells play an important role in orchestrating the effector function of CTLs in anti-tumour immunity. T-helper cell epitopes that trigger a T-helper cell response of the TH1 type support effector functions of CD8-positive killer T-cells, which include cytotoxic functions directed against tumour cells displaying tumour-associated peptide/MHC complexes on their cell surfaces. In this way tumour-associated T-helper cell epitopes, alone or in combination with other tumour-associated peptides, can serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumour immune responses.
Since both types of response, CD8 and CD4 dependent, contribute jointly and synergistically to the anti-tumour effect, the identification and characterization of tumour-associated antigens recognised by either CD8+ CTLs (ligand: MHC class I molecule+peptide epitope) or by CD4-positive CTLs (ligand: MHC class II molecule+peptide epitope) is important in the development of tumour vaccines. It is therefore an object of the present invention, to provide novel amino acid sequences for peptides that are able to bind to MHC complexes of either class.